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Protein complexes having factor VIII:C activity and production thereof

a technology of protein complexes and c activity, applied in the field of protein complexes having factor viii c activity, can solve the problems of difficult purification and characterization, and achieve the effects of high stability, high yield and high yield

Inactive Publication Date: 2006-06-08
CHIRON CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] We have now invented an improved method for expressing recombinant protein complexes with high stability and Factor VIII:C activity. The Mr 92 K polypeptide (FVIII-HC) and the Mr 80 K polypeptide (FVIII-LC) are expressed as two separate polypeptides, under the control of separate promoters, within the same host cell. Each polypeptide is preferably expressed using a signal sequence which directs export to the extracellular space with cleavage of the signal sequence. FVIII-HC is preferably expressed as a fusion protein having a C-terminal extension. The extension comprises a polypeptide sequence homologous to the B domain N-terminal sequence (which may allow cleavage by thrombin), a polypeptide spacer of 3 to 100 amino acids, and a sequence homologous to the C-terminal B domain sequence. The C-terminal extension of FVIII-HC results in a higher yield of active polypeptide upon expression in eukaryotic host cells. FVIII-LC is preferably expressed as an LC polypeptide using a signal peptide. The FVIII-LC polypeptide is processed and secreted efficiently with the correct N-terminal amino acid residue, and correct glycosylation. Co-transfection with polynucleotides encoding FVIII-HC and FVIII-LC in a suitable host cell provides recombinant protein complexes having Factor VIII:C activity in high yield.

Problems solved by technology

Although full-length recombinant human Factor VIII:C has been produced, it is difficult to purify and characterize, and it is unstable due to proteolysis.

Method used

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  • Protein complexes having factor VIII:C activity and production thereof
  • Protein complexes having factor VIII:C activity and production thereof
  • Protein complexes having factor VIII:C activity and production thereof

Examples

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example 1

Preparation of Expression Plasmids

[0045] (A) pSV7d:

[0046] The expression cassettes were prepared using the mammalian cell expression vector pSV7d (2423 bp).

[0047] The plasmid pSV7d (see Truett et al, supra) was constructed as follows: The 400 bp BamHI / HindIII fragment containing the SV40 origin of replication and early promoter was excised from pSVgtI (obtained from Paul Berg, Stanford University, California) and purified. The 240 bp SV40 BclI / BamHI fragment containing the SV40 polyA addition site was excised from pSV2 / DHFR (Subramani et al, Mol Cell Biol (1981) 1:854-864) and purified. The fragments were fused through the following linker:

This linker contains five restriction sites, as well as stop codons in all three reading frames. The resulting 670 bp fragment containing the SV40 origin of replication, the SV40 early promoter, the polylinker with stop codons and the SV40 polyadenylation site was cloned into the BamHI site of pML, a pBR322 derivative having about 1.5 Kb del...

example 2

[0076] Expression of the Mr 92 K protein in COS7 cells using the pSVF8-92 construction was low compared to the amount of Mr 80 K protein produced. The Mr 92 K protein is apparently retained and / or degraded in the Golgi pathway, and is not efficiently processed or exported. Accordingly, the construction was modified in an attempt to increase the level of Mr 92 K protein. Modifications of the following types were made: Changes in the 5′ untranslated sequence of the Factor VIII:C gene; inclusion of heterologous 5′ untranslated and leader sequences; and changes in the 3′ untranslated sequences. These constructs are summarized below.

[0077] (A) 5′ Untranslated Region Modifications Plasmid pSVF8-92B. This plasmid is a derivative of pSVF8-92 in which the 30 bp of 5′ untranslated sequence of pSVF8-92 is replaced with the entire 5′ untranslated region of human Factor VIII:C cDNA (nucleotides 1 to 171; see FIG. 8 of Truett et al, supra), with a deletion of the G-C tails (by in vitro site-spec...

example 3

[0087] This example describes the preparation of constructs for producing polypeptides that consist of the Mr 92 K chain and a portion of the B domain. These derivatives were made in an attempt to develop a heavy chain that is more stable and / or assembles more efficiently into an active complex with the light chain. The derivatives were chosen to mimic molecular species that have been observed in plasma-derived preparations of Factor VIII:C and in cell lysates and conditioned media from cells expressing recombinant full-length Factor VIII:C. Polypeptides of approximately the same size could possibly arise by thrombin cleavages of full-length Factor VIII:C.

[0088] (A) pSVF8-92S: This plasmid encodes a 982 amino acid heavy chain and was prepared from a full-length cDNA plasmid pSVF8-302 by cleavage at the first SacI site of the B-domain coding region. An oligonucleotide adaptor was used to install a translational stop codon and fuse the coding sequence to the natural human Factor VIII...

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Abstract

Recombinant protein complexes having human Factor VIII:C activity are expressed in a eukaryotic host cell by transforming the host cell with first and second expression cassettes encoding a first polypeptide substantially homologous to human Factor VIII:C A domain and a second polypeptide substantially homologous to human Factor VIII:C C domain, respectively. In the present invention, the first polypeptide may be extended having at its C-terminal a human Factor VIII:C B domain N-terminal peptide, a polypeptide spacer of 3-40 amino acids, and a human Factor VIII:C B domain C-terminal peptide. Expression of the second polypeptide is improved by employing an .alpha.sub.1-antitrypsin signal sequence.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of pending U.S. patent application Ser. No. 10 / 726,199, filed Dec. 1, 2003; which is a continuation of U.S. patent application Ser. No. 10 / 256,849, filed Sep. 26, 2002, now abandoned; which is a continuation of U.S. patent application Ser. No. 09 / 748,062, filed Dec. 22, 2000, now abandoned; which is a continuation of U.S. patent application Ser. No. 08 / 441,943, filed May 16, 1995, now U.S. Pat. No. 6,228,620; which is a division of U.S. patent application Ser. No. 08 / 161,770, filed Dec. 3, 1993, now U.S. Pat. No. 5,595,886; which is a continuation of U.S. patent application Ser. No. 07 / 652,099, filed Feb. 7, 1991, now abandoned; from which applications priority is claimed pursuant to 35 U.S.C. § 120, and all of which are incorporated herein by reference in their entirety.DESCRIPTION [0002] 1. Technical Field [0003] This invention relates to protein complexes having Factor VIII:C activity, and to method...

Claims

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Application Information

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IPC IPC(8): C12P21/04C12N5/06C12N5/08C07H21/04
CPCC07K14/755
Inventor CHAPMAN, BARBARABURKE, RAERASMUSSEN, MIRELLAMIKKELSON, JAN
Owner CHIRON CORP
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